Process Chromatography: Five Decades of Innovation

This article explores the development of process chromatography. Process chromatography was first applied to the removal of low molecular weight solutes from whey by gel filtration about 50 years ago. An analytical method using size exclusion chromatography was scaled up for insulin production in the 1970s, when ion exchange became a viable technology for the same application. Ion exchange was adopted as the industry workhorse as robust resins became available and formed the backbone of chromatographic processing of blood plasma fractionation in alternatives to and extensions of ethanol precipitation. Cost restrictions kept affinity chromatography in the laboratory until the production of MAbs made efficient immunoaffinity indispensable in high purity coagulation factor production in the 1980s. Since then, spurred on by the advent of biotechnology, an extensive toolbox of chromatographic methods has been developed, and a process chromatographic capture–purify– polish regime is ubiquitous. Affinity capture of antibodies on Protein A adsorbents is used throughout the industry with widespread discussion of affinity versus ion exchange. The emerging debate pitches chromatography against membrane separations. Column technology has advanced, but not to the "plug-and-play" status of membrane technologies. Axial flow systems still dominate, but advances in engineering may make radial flow accessible and technologies such as expanded beds more attractive. Process chromatography stands at the threshold of industrialization.

Professor Arne Tiselius, who had earlier described adsorption and displacement chromatography1 and later the use of hydroxyapatite, summed up the importance of partition chromatography in his presentation speech for the Nobel Prize in Chemistry (1952), awarded to Martin and Synge:

"This tool has enabled research workers in chemistry, biology, and medicine to tackle and solve problems which earlier were considered almost hopelessly complicated."2

It is perhaps the inherent simplicity of the method which has made chromatography not just an analytical tool par excellence but the central enabling technology in all biopharmaceutical downstream processing.

The work of these mid-century laureates has its roots in the investigations of Mikhail Tswett, who, although he described the principles of his separation techniques applied to plant pigments in 1903, first used the term chromatography in 1906:

"....the different components of a pigment mixture, obeying a law, are resolved on the calcium carbonate column and then can be qualitatively and quantitatively determined. I call such a preparation a chromatogram and the corresponding method the chromatographic method."3

The early history and invention of chromatography are summarized by Ettre in two articles in LCGC North America.3–4

THE 1960S: MATRICES, MOLECULES, AND ENGINEERING

Following Tswett's experimentation with various adsorbents and mobile phases, researchers in the 1950s investigated protein chromatography on new matrices. Low porosity, hydrophobic styrene-divinyl benzene resins were readily available, but for protein separations, porous and hydrophilic supports were needed. The introduction of cellulose ion exchangers by Peterson and Sobers in 1956,5 cross-linked dextrans (Sephadex) by Porath and Flodin in 1959,6 and polyacrylamide (1961) and agarose (1964) by Hjertén,7–8 initiated a revolution in protein chromatography. The first supports, generally referred to as "gels," were largely unsuitable for use in process chromatography: one gram of dry Sephadex G-100 adsorbs 100 mL of water and has therefore only 1% dry substance and 6% agarose media and 94% water.

Ligands and Matrices

The commercial availability of a range of carbohydrate-based supports enabled the expansion of chromatographic techniques. At this point, the science largely bifurcated into ligand discovery and matrix improvement. Axén's9 introduction of cyanogen bromide activation in 1967 allowed the development of affinity chromatography, the invention of which was attributed to Cuatrecasas et al. (1968).10 Interactions between Protein A and immunoglobulins were under investigation by Sjöqvist's11 group at Uppsala University in Sweden in the mid-1960s but IgG purification using Protein A adsorbents generally is ascribed to the Lund researchers Hjelm et al.12 and Kronvall et al.13 Uppsala, however, is intrinsically linked to bioseparations from the time of The Svedberg (Nobel Prize, 1926), through the activities of the Institute of Biochemistry (The Biomedical Centre) and the research and product development at Pharmacia Fine Chemicals, now part of GE Healthcare.

The drawbacks of hydrophobic Amberlite IRC-5014 and Dowex resins for protein separations gave rise to the search in the 1950s for matrices that did not interfere with the separation on derivatized gels. In 1947, Boscott15 had described the use of solvent-treated cellulose acetate as a "satisfactory stationary organic phase for chromatography," which Howard and Martin termed "reversed-phase partition chromatography" (RPC).16 RPC has a continuous polar stationary phase and requires organic solvents, whereas hydrophobic interaction chromatography (HIC) has polar ligands substituted onto a neutral backbone and is run with an aqueous mobile phase. Although these related technologies of RPC and HIC were born in the 1950s, they did not come to commercial use until considerably later.